The proposed research program is focused on an unusual mammal, the spotted hyena, as a route to understanding general mechanisms of sexual differentiation and development. The female spotted hyena has no external vagina. The clitoris has hypertrophied until it is the approximate size and shape of the male penis, and is traversed by a central urogenital canal. Female spotted hyenas urinate, mate and give birth through the clitoris. Contemporary understanding of sexual differentiation requires the presence of androgens during fetal life to explain the "masculine" phenotype observed at birth in both sexes. The present proposal, focused on differentiation of the urogenital system and the brain, is designed to examine the following hypotheses: (1) Differentiation and development of the external genitalia of the spotted hyena are driven, either by an androgen-independent process, or by "unusual" steroids (e.g., A and E2) activating the hyena Androgen Receptor (AR)). (2) Sex differences in clitoral/penile morphology observed at birth are the result of differential secretions of the fetal ovaries, testes or adrenals, acting on a background of placental T/E2 arriving via the umbilical vein. (3) Growth of the genitalia during postnatal life is modulated by the same growth factors that control general skeletal growth (i.e., IGF-1). (4) Sex differences in the brain are the result of sex differences in steroid hormones, such as those responsible for dimorphisms in the genitalia. In order to achieve these ends: (A) Dr. Michael McPhaul will extend his studies of the unusual binding properties of the hyena AR in fibroblast culture. In addition, a team of investigators will explore fetal urogenital development with a variety of techniques, including (B) transplantation of genital tissues from fetal hyenas into the renal capsule of nude mice; (C) examination of androgenic/estrogenic activity of fetal ovaries, testes and adrenals, including ICC studies of metabolic enzymes, and assays of gonadal secretions in vitro; and (D) examination of steroid receptors in the UG system during fetal life. (E) Studies of sex differences in the CNS, involving both Nissl stains and ICC for various neuropeptides, will examine fetal and adult tissues from the hyena CNS, including tissues derived from mature hyenas that were exposed to flutamide/finasteride in utero.